We previously demonstrated that the expression of two lipoproteins P7.5 and P35 Borrelia burgdorferi were upregulated at the onset of the stationary phase in cultured spirochetes. We expanded these studies to determine if there existed other proteins in B. burgdorferi that were also differentially expressed during in vitro growth. Spirochetes along the growth curve representing mid-logarithmic, late-logarithmic, early-stationary and late-stationary growth phases were harvested and processed for protein and RNA analyses. Proteins were analyzed by SDS-PAGE-silver staining and by Western blotting. The Western blots were developed either with 6 weeks post-infection serum from a B. burgdorferi-infected rhesus monkey, or with several monoclonal antibodies specific to previously characterized B. burgdorferi proteins. These studies revealed 9 additional antigens, including OspC, that were up-regulated 2-10-fold and 28 kDa protein that was down-regulated 2-fold, during the interval between the logarithmic and stationary growth phases. In contrast to these in vitro regulated proteins, the expression of OspA, OspB, GroEL, P72, flagellin, BmpA and BmpD, all remained unchanged throughout growth of the spirochetes in culture. ospAB, bmpAB, groEL and fla all exhibited similar mRNA profiles suggesting that these genes are constitutively expressed. On the other hand, bmpD exhibited a spike in RNA expression in early stationary phase, whereas ospC maintained a relatively higher level of RNA throughout culture as compared to the aforementioned constitutively expressed genes. These findings provide additional genes besides P7.5 and P35, whose regulated expression can be investigated in vitro in an otherwise intractable organism that cycles between an arthropod and a mammalian host.